CA2249181C - Scf analog compositions and methods - Google Patents
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- CA2249181C CA2249181C CA002249181A CA2249181A CA2249181C CA 2249181 C CA2249181 C CA 2249181C CA 002249181 A CA002249181 A CA 002249181A CA 2249181 A CA2249181 A CA 2249181A CA 2249181 C CA2249181 C CA 2249181C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention relates to stem cell factor ("SCF") analog polypeptide compositions, and vectors, host cells, and processes for recombinant DNA production of the present SCF analogs.
Description
SCF ANALOG COMPOSITIONS AND METHODS
FIELD OF THE INVENTION
The present invention relates to stem cell factor ("SCF") analog polypeptide compositions, and vectors, host cells, and processes for recombinant DNA
production of the present SCF analogs. Also, in yet other aspects, pharmaceutical compositions, and methods of use are provided.
BACKGROUND
Stem cell factor ("SCF", see PCT Pub. No.
WO 91/05795; also called kit-ligand, PCT Pub.
No. WO 92/03459, mast cell growth factor, see PCT Pub.
WO 92/00376 and Steel factor (or "SF" or "SLF") White, Cell 63: 5-6 (1990)) is hematopoietic factor which acts on hematopoietic progenitor cells. The gene encoding SCF has been cloned and expressed, e.g., Martin, et al., Cell 63: 203--211 (1990), and PCT Pub. No.WO 91/05795P
_ One hallmark of SCF activity is expansion of primitive progenitor cells in the marrow and.peripheral blbod. Clinically, SCF may prove useful in a number of rdisease settings where myelosuppression results in significant morbidity and mortality. These settings include the use of SCF in: mobilizing progenitor cells for use in transplantation following myeloablative chemotherapy, priming the marrow before bone-marrow haruest for autologous bone marrow transplantation, and in accelerating hematopoietic reconstitution following standard-dose chemotherapy or myeloablative chemotherapy/total body irradiatiori.
SCF has numerous active forms, including a membrane bound version and a soluble version. Sge PCT
FIELD OF THE INVENTION
The present invention relates to stem cell factor ("SCF") analog polypeptide compositions, and vectors, host cells, and processes for recombinant DNA
production of the present SCF analogs. Also, in yet other aspects, pharmaceutical compositions, and methods of use are provided.
BACKGROUND
Stem cell factor ("SCF", see PCT Pub. No.
WO 91/05795; also called kit-ligand, PCT Pub.
No. WO 92/03459, mast cell growth factor, see PCT Pub.
WO 92/00376 and Steel factor (or "SF" or "SLF") White, Cell 63: 5-6 (1990)) is hematopoietic factor which acts on hematopoietic progenitor cells. The gene encoding SCF has been cloned and expressed, e.g., Martin, et al., Cell 63: 203--211 (1990), and PCT Pub. No.WO 91/05795P
_ One hallmark of SCF activity is expansion of primitive progenitor cells in the marrow and.peripheral blbod. Clinically, SCF may prove useful in a number of rdisease settings where myelosuppression results in significant morbidity and mortality. These settings include the use of SCF in: mobilizing progenitor cells for use in transplantation following myeloablative chemotherapy, priming the marrow before bone-marrow haruest for autologous bone marrow transplantation, and in accelerating hematopoietic reconstitution following standard-dose chemotherapy or myeloablative chemotherapy/total body irradiatiori.
SCF has numerous active forms, including a membrane bound version and a soluble version. Sge PCT
Pub. No. 91/05795. C-terminal deletion analogs also have activity. For example, SCF 1-137 (with "1"
referring to the first amino acid of the mature protein) demonstrates biological activity, and SCF 1-141 demonstrates more or less full biological activity.
Langley et al., Arch. Biochem. Biophys. 311: 55-61 (1994). SCF 1-165 having an aspartic acid at position 10, instead of an asparagine as in the native sequence (referred to as "N10D") has also been studied, and found to not appreciably influence the rate of dimer formation. Lu et al., Biochem. J. 305: 563-568 (1995).
Certain covalently linked SCF dimers are reported in PCT
publication WO 95/26199. Stability is reportedly increased by an intermolecular covalent linkages.
SCF analogs with increased biological activity and stability, such as those provided herein, would be desirable for consumers, as lower dosages may be used to achieve the same biological result. Such analogs would be desirable for producers, as a more active product results in more units of product being sold per production batch. Increased stability, particularly increased shelf-life, would be particularly useful for both consumers and producers. Thus, the present invention provides these advantages and satisfies the needs of consumers and producers.
SUMMARY OF THE INVENTION
The present invention relates to SCF analogs containing aspartic acid substituted for asparagine at position 10 and position 11 (according to the numbering of FIGURE 1, where methionine is at position -1).
Surprisingly, and importantly, it has been found that these substitutions result in SCF analogs having substantially higher biological activity and increased stability as compared to unmodified SCF.
referring to the first amino acid of the mature protein) demonstrates biological activity, and SCF 1-141 demonstrates more or less full biological activity.
Langley et al., Arch. Biochem. Biophys. 311: 55-61 (1994). SCF 1-165 having an aspartic acid at position 10, instead of an asparagine as in the native sequence (referred to as "N10D") has also been studied, and found to not appreciably influence the rate of dimer formation. Lu et al., Biochem. J. 305: 563-568 (1995).
Certain covalently linked SCF dimers are reported in PCT
publication WO 95/26199. Stability is reportedly increased by an intermolecular covalent linkages.
SCF analogs with increased biological activity and stability, such as those provided herein, would be desirable for consumers, as lower dosages may be used to achieve the same biological result. Such analogs would be desirable for producers, as a more active product results in more units of product being sold per production batch. Increased stability, particularly increased shelf-life, would be particularly useful for both consumers and producers. Thus, the present invention provides these advantages and satisfies the needs of consumers and producers.
SUMMARY OF THE INVENTION
The present invention relates to SCF analogs containing aspartic acid substituted for asparagine at position 10 and position 11 (according to the numbering of FIGURE 1, where methionine is at position -1).
Surprisingly, and importantly, it has been found that these substitutions result in SCF analogs having substantially higher biological activity and increased stability as compared to unmodified SCF.
The present analogs resulted in part from observations made during in vitro aging studies, where wild type SCF was found to be highly unstable in certain formulation buffers. Such wild type SCF would be predicted to have shorter shelf life. It has now been found that the major instability of wild type SCF is caused by the deamidation reaction at aspartyl (asparagine, ASN, "N") residue at sequence position 10 (with respect to the mature protein as in Figure 1).
Deamidation also resulted in isomerization of the deamidated aspartyl residue by alpha/beta peptide bond shift. The isomerized SCF species is not biologically active. There is also a risk of antigenicity due to subtle change in the molecular structure. As demonstrated below, the analogs provided herein are more stable than wild type SCF, have increased biological activity, and eliminate any risk associated with deamidation.
Other aspects of the present invention include nucleic acids encoding for the present SCF analogs, vectors, host cells, and processes for recombinant DNA
production of the present SCF analogs. Also, in yet other aspects, pharmaceutical compositions, and methods of use are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is an illustration of the amino acid sequence for amino acids 1-248 of the membrane bound form of human recombinant SCF.
FIGURE 2 is a graph illustrating the relative amount of intact protein/analog in an accelerated stability assay (see infra). The samples were maintained at 37 C for 1-5 days, and then analyzed for degradation products.
The samples tested were (a)"rhSCF, wild type", recombinant human met-1 SCF 1-165, (b) "N10D, N11D
variant", recombinant human met-1 SCF 1-165 having an aspartic acid residue at positions 10 and 11; (c) "N10D
variant", i.e., the same as (b), above, except with the aspartic acid at position 10 only; and (d) "N10E
variant" the same as N10D except with a glutamic acid at position 10.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to particular SCF analogs demonstrating higher biological activity than unmodified SCF. As indicated above, it has been found that substitution of one or both of the asparagines at positions 10 and 11 (according to FIGURE
1) results in a molecule demonstrating substantially higher biological activity than unmodified SCF. The present analogs are herein referred to as N10D (for asparagine at the 10th position substituted with asparatic acid) and N10D, N11D (for the SCF analog having an additional substitution at the llth position).
Preferred is N10D, N11D, as changes at both locations result in additional biological activity (in a megakaryocytic cell proliferation assay) over the change N10D alone.
For convenience, the term "N10D" and "N10D, N11D" refer generically to the present SCF analogs, whether analogs of the full length 1-248 SCF protein (FIGURE 1) or analogs of some other form as described below. When discussing a particular analog herein, the particular unmodified amino acid sequence is referred to. For example, demonstrated below is SCF analog N10D
SCF met 1-165 (an analog of SCF having 165 amino acids of FIGURE 1 with an N-terminal methionyl residue, and aspartic acid substituted for asparagine at the 10th position).
Deamidation also resulted in isomerization of the deamidated aspartyl residue by alpha/beta peptide bond shift. The isomerized SCF species is not biologically active. There is also a risk of antigenicity due to subtle change in the molecular structure. As demonstrated below, the analogs provided herein are more stable than wild type SCF, have increased biological activity, and eliminate any risk associated with deamidation.
Other aspects of the present invention include nucleic acids encoding for the present SCF analogs, vectors, host cells, and processes for recombinant DNA
production of the present SCF analogs. Also, in yet other aspects, pharmaceutical compositions, and methods of use are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is an illustration of the amino acid sequence for amino acids 1-248 of the membrane bound form of human recombinant SCF.
FIGURE 2 is a graph illustrating the relative amount of intact protein/analog in an accelerated stability assay (see infra). The samples were maintained at 37 C for 1-5 days, and then analyzed for degradation products.
The samples tested were (a)"rhSCF, wild type", recombinant human met-1 SCF 1-165, (b) "N10D, N11D
variant", recombinant human met-1 SCF 1-165 having an aspartic acid residue at positions 10 and 11; (c) "N10D
variant", i.e., the same as (b), above, except with the aspartic acid at position 10 only; and (d) "N10E
variant" the same as N10D except with a glutamic acid at position 10.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to particular SCF analogs demonstrating higher biological activity than unmodified SCF. As indicated above, it has been found that substitution of one or both of the asparagines at positions 10 and 11 (according to FIGURE
1) results in a molecule demonstrating substantially higher biological activity than unmodified SCF. The present analogs are herein referred to as N10D (for asparagine at the 10th position substituted with asparatic acid) and N10D, N11D (for the SCF analog having an additional substitution at the llth position).
Preferred is N10D, N11D, as changes at both locations result in additional biological activity (in a megakaryocytic cell proliferation assay) over the change N10D alone.
For convenience, the term "N10D" and "N10D, N11D" refer generically to the present SCF analogs, whether analogs of the full length 1-248 SCF protein (FIGURE 1) or analogs of some other form as described below. When discussing a particular analog herein, the particular unmodified amino acid sequence is referred to. For example, demonstrated below is SCF analog N10D
SCF met 1-165 (an analog of SCF having 165 amino acids of FIGURE 1 with an N-terminal methionyl residue, and aspartic acid substituted for asparagine at the 10th position).
The SCF analogs may be analogs as compared to the full length molecule of human SCF (as in FIGURE 1) or may be analogs of a modified SCF, such as SCF having amino acids 1-165 or 1-164 of FIGURE 1. (The present working examples demonstrate modified SCF 1-165 having a methionyl residue at position -1). Another type of SCF
is one having 220 amino acids, due to the deletion of an exon (the membrane bound version of SCF). ee, PCT WO
91/05795, entitled, "Stem Cell Factor," published May 2, 1991,.
The above Stem Cell Factor PCT publication, at Figure 44, illustrates SCF 1-220. Modified SCFs also include those with deletions of one or more amino acids at the C-terminus, down to amino acids 1-138, or more preferably, 1-141 (of FIGURE 1), and those with N-terminal deletions of position 1 through 4 (also of FIGURE 1).
Modified SCF may include those having other additions, deletions or substitutions, so long as they possess at least one of the hematopoietic biological activities of naturally occurring human SCF. A preferred hematopoietic biological activity is the ability to stimulate the growth of early hematopoietic progenitor cells. See the SCF PCT publication WO 91/05795, passim. Modification using the present invention of substitution of the asparagine to aspartic acid at the corresponding site on the "starting" SCF molecule is expected to increase the activity of the "starting" SCF
molecule. (The term "starting" is quoted because, typically, the nucleic acid encoding these proteins, and not the protein itself, is the starting point for producing the present analogs.) Specific modified SCFs which may be used as "starting" material to practice the present invention, (as changes corresponding to N10D and N10D, N11D are likely to increase biological activity) include (with reference to FIGURE 1):
(a) any of SCF 1-138 or, preferably 1-141 (at least having the C-terminal cysteine of FIGURE 1 at that position), through 1-248, as these C-terminal deletions do not appreciably affect biological activity in a UT-7 bioassay (as described below);
(b) SCF 1-127, 1-130 and 1-137, having reduced activity; and (c) any of the above SCFs having N-terminal deletions at the 1, 2, 3, or 4 positions (and having the cysteine remaining at position 4).
In addition, the covalent dimers as reported in WO 95/26199, such as those in which amino acid linkers are used to covalently link the N-terminus of one monomer to the C-terminus of another monomer may include one or more monomer which is an analog of the present invention.
Preferred for "starting" SCFs are SCF 1-248, SCF 1-165, SCF 1-164, as these are thought to possess the greatest biological activity, and have been characterized.
The present SCF analogs may have a methionyl residue at position -1 (typically incident to bacterial expression).
The present SCF analogs may be included in a preparation of monomeric or dimeric forms, and homodimers are preferred for maintaining the highest biological activity. One may have a heterodimer comprised of one N10D and one N10D, N11D molecule, also with heightened activity. One may also prepare a heterodimer with N10D, N11D and native SCF (such as unmodified SCF 1-164 or 1-165). The SCF preparation may be a mixture of hetero- and homo-dimers, or may be a mixture of such dimers and monomers.
is one having 220 amino acids, due to the deletion of an exon (the membrane bound version of SCF). ee, PCT WO
91/05795, entitled, "Stem Cell Factor," published May 2, 1991,.
The above Stem Cell Factor PCT publication, at Figure 44, illustrates SCF 1-220. Modified SCFs also include those with deletions of one or more amino acids at the C-terminus, down to amino acids 1-138, or more preferably, 1-141 (of FIGURE 1), and those with N-terminal deletions of position 1 through 4 (also of FIGURE 1).
Modified SCF may include those having other additions, deletions or substitutions, so long as they possess at least one of the hematopoietic biological activities of naturally occurring human SCF. A preferred hematopoietic biological activity is the ability to stimulate the growth of early hematopoietic progenitor cells. See the SCF PCT publication WO 91/05795, passim. Modification using the present invention of substitution of the asparagine to aspartic acid at the corresponding site on the "starting" SCF molecule is expected to increase the activity of the "starting" SCF
molecule. (The term "starting" is quoted because, typically, the nucleic acid encoding these proteins, and not the protein itself, is the starting point for producing the present analogs.) Specific modified SCFs which may be used as "starting" material to practice the present invention, (as changes corresponding to N10D and N10D, N11D are likely to increase biological activity) include (with reference to FIGURE 1):
(a) any of SCF 1-138 or, preferably 1-141 (at least having the C-terminal cysteine of FIGURE 1 at that position), through 1-248, as these C-terminal deletions do not appreciably affect biological activity in a UT-7 bioassay (as described below);
(b) SCF 1-127, 1-130 and 1-137, having reduced activity; and (c) any of the above SCFs having N-terminal deletions at the 1, 2, 3, or 4 positions (and having the cysteine remaining at position 4).
In addition, the covalent dimers as reported in WO 95/26199, such as those in which amino acid linkers are used to covalently link the N-terminus of one monomer to the C-terminus of another monomer may include one or more monomer which is an analog of the present invention.
Preferred for "starting" SCFs are SCF 1-248, SCF 1-165, SCF 1-164, as these are thought to possess the greatest biological activity, and have been characterized.
The present SCF analogs may have a methionyl residue at position -1 (typically incident to bacterial expression).
The present SCF analogs may be included in a preparation of monomeric or dimeric forms, and homodimers are preferred for maintaining the highest biological activity. One may have a heterodimer comprised of one N10D and one N10D, N11D molecule, also with heightened activity. One may also prepare a heterodimer with N10D, N11D and native SCF (such as unmodified SCF 1-164 or 1-165). The SCF preparation may be a mixture of hetero- and homo-dimers, or may be a mixture of such dimers and monomers.
Novel nucleic acid sequences of the invention include sequences useful in securing expression in procaryotic or eucaryotic host cells of the present SCF
analogs, N10D and N10D, N11D. The nucleic acids may be purified and isolated, so that the desired coding region is useful to produce the present polypeptides.
More specifically, DNA sequences of the invention comprise:
(a) the DNA sequence set forth in Sequence ID Nos. 3 and 4, and 6 and 7;
(b) the DNA sequences of subpart (a) modified to encode another version of SCF having at least one of the hematopoietic biological properties of naturally occurring human SCF. Preferably the biological property is the property of binding to an SCF
receptor (the c-kit receptor), but another biological properties is capability of stimulation of the proliferation of early hematopoietic progenitor cells.
Another biological property is capability of stimulation of melanocytes to produce melanin. Other biological properties will be apparent to those skilled in the art.
More specifically, the present nucleic acids are those encoding N10D or N10D, N11D in (with reference to FIGURE 1):
(a) any of SCF 1-138 or, preferably 1-141 (at least having the C-terminal cysteine of FIGURE 1 at that position), through 1-248;
(b) SCF 1-127, 1-130 and 1-137; and (c) any of the above SCFs having N-terminal deletions at the 1, 2, 3, or 4 positions (and having the cysteine remaining at position 4).
DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in microbial hosts. Such manufactured sequences may readily be constructed according to the methods of Alton et al., PCT published application WO 83/04053. The DNAs may optionally encode an N-terminal methionyl residue.
DNA sequences provided by the invention are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of expression of the present SCF analogs. The DNA sequences encoding biologically active SCF analogs provided herein (or corresponding RNAs) may be useful for gene therapy in instances where underproduction of SCF would be alleviated.
The present invention also provides for processes for production of the present SCF analogs.
Provided is a process for producing SCF analog N10D
and/or N10D, N11D, from a host cell containing nucleic acid encoding such SCF analog comprised of:
(a) culturing said host cell containing nucleic acid encoding such SCF analog under conditions facilitating the expression of such DNA molecule; and (b) obtaining such SCF analog.
One may optionally purify and isolate such SCF analog from other components obtained in the process.
Host cells may be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary cells, monkey cells, baby hamster kidney cells, or other cells), yeast cells, and insect cells.
Preferred for greatest ease in commercial production is production using a bacterial host cell.
Also comprehended by the invention are pharmaceutical compositions comprising effective amounts of polypeptide products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in SCF therapy. Such compositions include diluents of various buffer content (e.g., Tris-HC1, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween*
80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.q., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or in association with liposomes. Such compositions will influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present SCF analogs. ee, e.a., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712.
Derivatives of the present SCF analogs are also comprehended herein. Such derivatives include molecules modified by one or more water soluble polymer molecules, such as polyethylene glycol, or by the addition of polyamino acids. Such derivatization may occur singularly at the N- or C-terminus or there may be multiple sites of derivatization. Substitution of one or more amino acids with lysine may provide additional sites for derivatization.
r The present analogs or derivatives thereof may be formulated for injection, or oral, nasal, pulmonary, topical, or other types of administration as one skilled in the art will recognize. The formulation may be liquid or mlay be solid, such as lyophilized, for reconstitution.
In general, the present analogs (or derivatives thereof) will be useful in the same way that currently available SCFs are useful, except the present analogs provide for greater efficacy (approximately 200%
as will be demonstrated below) 'These uses include the * Trade-mark treatment of a variety of hematopoietic, neurological and reproduction related conditions. See WO 91/05795 see also US Patent 6,204,363.
Thus, the present compositions and methods for manufacture of medicaments may be useful for the treatment of such conditions. Such conditions include but are not limited to the treatment of leucopenia, the treatment of thrombocytopenia, the treatment of anemia, enhancing engraftment of bone marrow during transplantation, enhancing bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, acquired immune deficiency syndrome, and sensitizing cells to chemotherapy. In addition, the present SCF
analog compositions may be used for mobilization of peripheral blood progenitor cells. Such uses and compositions also include providing treatment to a mammal suffering from nerve damage, infertility, or intestinal damage. In addition, the present SCF analog compositions may be useful for the treatment of pigmentation disorders, such as vitilago.
The present SCF analog (or derivative) compositions may also be used in vitro. For example, in a gene therapy setting, one may desire to transfect a hematopoietic cell with exogenous DNA, and culture said cell using the present SCF analog formulations. Thus, in yet another aspect the present invention involves a method for culturing hematopoietic cells in vitro comprised of:
(i) placing said cells in a suitable culture medium, said suitable culture medium containing an SCF analog composition according to the present invention, and (ii) providing suitable conditions for the growth of said hematopoietic cells.
analogs, N10D and N10D, N11D. The nucleic acids may be purified and isolated, so that the desired coding region is useful to produce the present polypeptides.
More specifically, DNA sequences of the invention comprise:
(a) the DNA sequence set forth in Sequence ID Nos. 3 and 4, and 6 and 7;
(b) the DNA sequences of subpart (a) modified to encode another version of SCF having at least one of the hematopoietic biological properties of naturally occurring human SCF. Preferably the biological property is the property of binding to an SCF
receptor (the c-kit receptor), but another biological properties is capability of stimulation of the proliferation of early hematopoietic progenitor cells.
Another biological property is capability of stimulation of melanocytes to produce melanin. Other biological properties will be apparent to those skilled in the art.
More specifically, the present nucleic acids are those encoding N10D or N10D, N11D in (with reference to FIGURE 1):
(a) any of SCF 1-138 or, preferably 1-141 (at least having the C-terminal cysteine of FIGURE 1 at that position), through 1-248;
(b) SCF 1-127, 1-130 and 1-137; and (c) any of the above SCFs having N-terminal deletions at the 1, 2, 3, or 4 positions (and having the cysteine remaining at position 4).
DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in microbial hosts. Such manufactured sequences may readily be constructed according to the methods of Alton et al., PCT published application WO 83/04053. The DNAs may optionally encode an N-terminal methionyl residue.
DNA sequences provided by the invention are useful in generating new and useful viral and plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of expression of the present SCF analogs. The DNA sequences encoding biologically active SCF analogs provided herein (or corresponding RNAs) may be useful for gene therapy in instances where underproduction of SCF would be alleviated.
The present invention also provides for processes for production of the present SCF analogs.
Provided is a process for producing SCF analog N10D
and/or N10D, N11D, from a host cell containing nucleic acid encoding such SCF analog comprised of:
(a) culturing said host cell containing nucleic acid encoding such SCF analog under conditions facilitating the expression of such DNA molecule; and (b) obtaining such SCF analog.
One may optionally purify and isolate such SCF analog from other components obtained in the process.
Host cells may be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese Hamster Ovary cells, monkey cells, baby hamster kidney cells, or other cells), yeast cells, and insect cells.
Preferred for greatest ease in commercial production is production using a bacterial host cell.
Also comprehended by the invention are pharmaceutical compositions comprising effective amounts of polypeptide products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in SCF therapy. Such compositions include diluents of various buffer content (e.g., Tris-HC1, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween*
80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.q., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or in association with liposomes. Such compositions will influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present SCF analogs. ee, e.a., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712.
Derivatives of the present SCF analogs are also comprehended herein. Such derivatives include molecules modified by one or more water soluble polymer molecules, such as polyethylene glycol, or by the addition of polyamino acids. Such derivatization may occur singularly at the N- or C-terminus or there may be multiple sites of derivatization. Substitution of one or more amino acids with lysine may provide additional sites for derivatization.
r The present analogs or derivatives thereof may be formulated for injection, or oral, nasal, pulmonary, topical, or other types of administration as one skilled in the art will recognize. The formulation may be liquid or mlay be solid, such as lyophilized, for reconstitution.
In general, the present analogs (or derivatives thereof) will be useful in the same way that currently available SCFs are useful, except the present analogs provide for greater efficacy (approximately 200%
as will be demonstrated below) 'These uses include the * Trade-mark treatment of a variety of hematopoietic, neurological and reproduction related conditions. See WO 91/05795 see also US Patent 6,204,363.
Thus, the present compositions and methods for manufacture of medicaments may be useful for the treatment of such conditions. Such conditions include but are not limited to the treatment of leucopenia, the treatment of thrombocytopenia, the treatment of anemia, enhancing engraftment of bone marrow during transplantation, enhancing bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, acquired immune deficiency syndrome, and sensitizing cells to chemotherapy. In addition, the present SCF
analog compositions may be used for mobilization of peripheral blood progenitor cells. Such uses and compositions also include providing treatment to a mammal suffering from nerve damage, infertility, or intestinal damage. In addition, the present SCF analog compositions may be useful for the treatment of pigmentation disorders, such as vitilago.
The present SCF analog (or derivative) compositions may also be used in vitro. For example, in a gene therapy setting, one may desire to transfect a hematopoietic cell with exogenous DNA, and culture said cell using the present SCF analog formulations. Thus, in yet another aspect the present invention involves a method for culturing hematopoietic cells in vitro comprised of:
(i) placing said cells in a suitable culture medium, said suitable culture medium containing an SCF analog composition according to the present invention, and (ii) providing suitable conditions for the growth of said hematopoietic cells.
More particularly, the present invention provides a method of transfecting hematopoietic cells with exogenous DNA comprising:
(i) culturing said hematopoietic cells with an SCF analog composition according to the present invention, and (ii) transfecting said cultured cell with exogenous DNA. The hematopoietic cells may be, for example, bone marrow cells or peripheral blood progenitor cells.
In yet another aspect, the present invention provides a kit containing components for culturing bone marrow cells or peripheral blood progenitor cells comprised of:
(i) an SCF analog composition of the present invention; and (ii) components suitable for preparing medium for culturing bone marrow cells or peripheral blood progenitor cells.
The uses or products herein may involve the administration or inclusion of at least one additional factor selected from among EPO, G-CSF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IGF-1, LIF, interferon (such as (c, (3, gamma or consensus) neurotrophic factors (such as BDNF, NT-3, CTNF or noggin), other multi-potent growth factors (such as, to the extent these are demonstrated to be such multi-potent growth factor, flt-3/flk-2 ligand, stem cell proliferation factor, and totipotent stem cell factor), fibroblast growth factors (such as FGF), and analogs, fusion molecules or other derivatives of the above. For example, SCF in combination with G-CSF has been found to mobilize peripheral blood progenitor cells in v' o. Ex vivo, for example, SCF in combination with G-CSF, IL-3 and IL-6 has been found useful for expansion of peripheral blood cells. The present analogs will provide for similar uses.
Generally, an effective amount of the present SCF analogs (or derivatives) will be determined by the age, weight and condition or severity of disease of the recipient. SPe, Remingtons Pharmaceutical Sciences, su.ra, at pages 697-773, Typically, a dosage of between about 0.001 g/kg body weight/day to about 1000 g/kg body weight/day, may be used, but more or less, as a skilled practitioner will recognize, may be used. Dosing may be one or more times daily, or less frequently, and may be in conjunction with other compositions as described herein. It should be noted that the present invention is not limited to the dosages recited herein.
The following example are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof. The example illustrates the preparation of the present SCF met 1-165 N10D and N10D, N11D, and the determination that avoiding deamidation at the asparagine at the 10th and/or llth 'position avoids degradation by-products of recombinant human SCF. The testing of these SCF analogs in vitro is also described.
EXAMPLES
1. Determination that the asparagine residue at positions 10 and/or 11 influences the biological function and chemical stabilitv of recombinant human SCF=
During in vitro aging, deamidation of recombinant human stem cell factor (rhSCF1-165) was detected by HPLC analysis and by release of soluble ammonia. Deamidation rate is slow in buffers at a low pH or at low temperatures, but the rate is significantly accelerated in alkaline bufers such as sodium bicarbonate in combination with elevated temperatures.
HPLC isolation of various deamidated forms followed by peptide map analysis revealed that the deamidation involved asparagine at the 10th position in protein.
Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of the asparagine at position 10 into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of the rhSCF dimer (see, Lu et al., Biochem J. 305: 563-568 (1995)), a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl rhSCF and other rhSCF monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of asparagine is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis as described above, had higher activity than the standard molecule. In comparison, synthetic N10A
(having an alanine at the 10th position) or N10E (having a glutamic acid at the 10th position), though missing the deamidation site, were significantly less active.
All these analogs were relatively more stable than those containing the asparagine residue. These results indicated that the biological function and the chemical stability of recombinant human SCF are influenced by the nature of the residue at position 10.
(i) culturing said hematopoietic cells with an SCF analog composition according to the present invention, and (ii) transfecting said cultured cell with exogenous DNA. The hematopoietic cells may be, for example, bone marrow cells or peripheral blood progenitor cells.
In yet another aspect, the present invention provides a kit containing components for culturing bone marrow cells or peripheral blood progenitor cells comprised of:
(i) an SCF analog composition of the present invention; and (ii) components suitable for preparing medium for culturing bone marrow cells or peripheral blood progenitor cells.
The uses or products herein may involve the administration or inclusion of at least one additional factor selected from among EPO, G-CSF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IGF-1, LIF, interferon (such as (c, (3, gamma or consensus) neurotrophic factors (such as BDNF, NT-3, CTNF or noggin), other multi-potent growth factors (such as, to the extent these are demonstrated to be such multi-potent growth factor, flt-3/flk-2 ligand, stem cell proliferation factor, and totipotent stem cell factor), fibroblast growth factors (such as FGF), and analogs, fusion molecules or other derivatives of the above. For example, SCF in combination with G-CSF has been found to mobilize peripheral blood progenitor cells in v' o. Ex vivo, for example, SCF in combination with G-CSF, IL-3 and IL-6 has been found useful for expansion of peripheral blood cells. The present analogs will provide for similar uses.
Generally, an effective amount of the present SCF analogs (or derivatives) will be determined by the age, weight and condition or severity of disease of the recipient. SPe, Remingtons Pharmaceutical Sciences, su.ra, at pages 697-773, Typically, a dosage of between about 0.001 g/kg body weight/day to about 1000 g/kg body weight/day, may be used, but more or less, as a skilled practitioner will recognize, may be used. Dosing may be one or more times daily, or less frequently, and may be in conjunction with other compositions as described herein. It should be noted that the present invention is not limited to the dosages recited herein.
The following example are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof. The example illustrates the preparation of the present SCF met 1-165 N10D and N10D, N11D, and the determination that avoiding deamidation at the asparagine at the 10th and/or llth 'position avoids degradation by-products of recombinant human SCF. The testing of these SCF analogs in vitro is also described.
EXAMPLES
1. Determination that the asparagine residue at positions 10 and/or 11 influences the biological function and chemical stabilitv of recombinant human SCF=
During in vitro aging, deamidation of recombinant human stem cell factor (rhSCF1-165) was detected by HPLC analysis and by release of soluble ammonia. Deamidation rate is slow in buffers at a low pH or at low temperatures, but the rate is significantly accelerated in alkaline bufers such as sodium bicarbonate in combination with elevated temperatures.
HPLC isolation of various deamidated forms followed by peptide map analysis revealed that the deamidation involved asparagine at the 10th position in protein.
Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of the asparagine at position 10 into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of the rhSCF dimer (see, Lu et al., Biochem J. 305: 563-568 (1995)), a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl rhSCF and other rhSCF monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of asparagine is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis as described above, had higher activity than the standard molecule. In comparison, synthetic N10A
(having an alanine at the 10th position) or N10E (having a glutamic acid at the 10th position), though missing the deamidation site, were significantly less active.
All these analogs were relatively more stable than those containing the asparagine residue. These results indicated that the biological function and the chemical stability of recombinant human SCF are influenced by the nature of the residue at position 10.
2. Preparation of SCF met 1-165 N10D,and N10D, In view of the above studies demonstrating that deamidation at position 10 resulted in loss of activity and instability of rhSCF preparations, the present N10D and N10D, N11D analogs were prepared and tested for biological activity and stability.
The present SCF analogs were prepared by site-directed mutagenesis of SCF met 1-165. The E. coli expression of SCF met 1-165 was previously reported.
Langley et al., Arch. Biochem. Biophys. 295: 21-28 (1992). The DNA encoding recombinant human SCF had an initial methionine codon followed by codons for human SCF 1-165. The purified recombinant human SCF retains the initiating Met (position Met -1). The present N10D
and N10D, N11D analogs were prepared by site-directed mutagenesis as described in Langley et al., Arch.
Biochem. Biophys. 311: 55-61 (1994). Other "test"
analogs, N10A and N10E, were also prepared in this fashion.
The present SCF analogs were prepared by site-directed mutagenesis of SCF met 1-165. The E. coli expression of SCF met 1-165 was previously reported.
Langley et al., Arch. Biochem. Biophys. 295: 21-28 (1992). The DNA encoding recombinant human SCF had an initial methionine codon followed by codons for human SCF 1-165. The purified recombinant human SCF retains the initiating Met (position Met -1). The present N10D
and N10D, N11D analogs were prepared by site-directed mutagenesis as described in Langley et al., Arch.
Biochem. Biophys. 311: 55-61 (1994). Other "test"
analogs, N10A and N10E, were also prepared in this fashion.
The amino acid sequence and nucleic acid sequence for SCF met 1--1651 _ was (Seq. ID Nos. 1 (DNA) and 2 (amino acid)):
ATGGAAGGTA TCTGCCGTAA CCGTGTTACT AACAACGTTA AAGACGTTAC
M E G I C R N R V T N N V K D V T
TAAACTGGTT GCTAACCTGC CGAAAGACTA CATGATCACC CTGAAATACG
K L V A N L P K D Y M I T L K Y
TTCCGGGTAT GGACGTTCTG CCGTCTCACT GCTGGATCTC TGAAATGGTT
V P G M D V L P S H C W I S E M V
GTTCAGCTGT CTGACTCTCT GACTGACCTG CTGGACAAAT TCTCTAACAT
V Q L S D S L T D L L D K F S N I
CTCTGAAGGT CTGTCTAACT ACTCTATCAT CGACAAACTG GTTAACATCG
S E G L S N Y S I I D K L V N I
TTGACGACCT GGTTGAATGC GTTAAAGAAA ACTCTTCTAA AGACCTGAAA
V D D L V E C: V K E N S S :K D L K
AAATCTTTCA AATCTCCGGA ACCGCGTCTG TTCACTCCGG AAGAATTCTT
K S F K S P E P R L F T P E E F F
CCGTATCTTC AACCGTTCCA TCGACGCTTT CAAAGACTTC GTTGTTGCTT
R I F N R S I D A F K D F V V A
CCGAAACCTC CGACTGCGTT GTTTCCTCCA CCCTGTCTCC GGAAAAAGAC
S E T S D C V V S S T L S P E K D
TCCCGTGTTT CGGTTACCAA ACCGTTCATG CTGCCGCCGG TTGCTGCTTAA
S R V S V T K P F M L P P V A A *
r The amino acid sequence and nucleic acid sequence for SCF met 1-165 N10D was (Seq. ID Nos. 3 and 4 (DNA) and 5 (amino acid)):
CATATGGAAGGTATCTGCAGGAATCGTGTGACTGACAATGTAAAAGACGTCACTAAATTG
1 ---------+---------+---------+---------+---------+---------+ 60 GTATACCTTCCATAGACGTCCTTAGCACACTGACTGTTACATTTTCTGCAGTGATTTAAC
M E G I C R N R V T ]2 N V K D V T K L
GTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTT
61 ---------+---------+---------+---------+---------+---------+ 120 CACCGTTTAGAAGGTTTTCTGATGTACTATTGGGAGTTTATACAGGGGCCCTACCTACAA
V A N L P K D Y M I T L K Y V P G M D V
TTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGAT
121 ---------+---------+---------+---------+---------+---------+ 180 AACGGTTCAGTAACAACCTATTCGCTCTACCATCATGTTAACAGTCTGTCGAACTGACTA
L P S H C W I S E M V V Q L S D S L T D
CTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAA
181 ---------+---------+---------+---------+---------+---------+ 240 GAAGACCTGTTCAAAAGTTTATAAAGACTTCCGAACTCATTAATAAGGTAGTATCTGTTT
L L D K F S N I S E G L S N Y S I I D K
CTTGTGAATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTA
241 ---------+---------+---------+---------+---------+---------+ 300 GAACACTTATATCACCTACTGGAACACCTCACGCACTTTCTTTTGP,GTAGATTCCTAGAT
L V N I V D D L V E C V K E N S S K D L
AAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTCCGTATC
301 ---------+---------+---------+---------+---------+---------+ 360 TTTTTTAGTAAGTTCTCGGGTCTTGGGTCCGAGAAATGAGGACTTCTTAAGAAGGCATAG
K K S F K S P E P R L F T P E E F F R I
TTCAACCGTTCCATCGACGCTTTCAAAGACTTCGTTGTTGCTTCCGAAACCTCCGACTGC
361 ---------+---------+---------+---------+---------+---------+ 420 AAGTTGGCAAGGTAGCTGCGAAAGTTTCTGAAGCAACAACGAAGGCTTTGGAGGCTGACG
F N R S I D A F K D F V V A S E T S D C
GTTGTTTCCTCCACCCTGTCTCCGGAAAAAGACTCCCGTGTTTCGGTTACCAAACCGTTC
421 ---------+---------+---------+---------+---------+---------+ 480 CAACAAAGGAGGTGGGACAGAGGCCTTTTTCTGAGGGCACAAAGCCAATGGTTTGGCAAG
V V S S T L S P E K D S R V S V T K P F
ATGCTGCCGCCGGTTGCTGCTTAATAGGATCC
481 ---------+---------+---------+-- 512 TACGACGGCGGCCAACGACGAATTATCCTAGG
M L P P V A A * *
ATGGAAGGTA TCTGCCGTAA CCGTGTTACT AACAACGTTA AAGACGTTAC
M E G I C R N R V T N N V K D V T
TAAACTGGTT GCTAACCTGC CGAAAGACTA CATGATCACC CTGAAATACG
K L V A N L P K D Y M I T L K Y
TTCCGGGTAT GGACGTTCTG CCGTCTCACT GCTGGATCTC TGAAATGGTT
V P G M D V L P S H C W I S E M V
GTTCAGCTGT CTGACTCTCT GACTGACCTG CTGGACAAAT TCTCTAACAT
V Q L S D S L T D L L D K F S N I
CTCTGAAGGT CTGTCTAACT ACTCTATCAT CGACAAACTG GTTAACATCG
S E G L S N Y S I I D K L V N I
TTGACGACCT GGTTGAATGC GTTAAAGAAA ACTCTTCTAA AGACCTGAAA
V D D L V E C: V K E N S S :K D L K
AAATCTTTCA AATCTCCGGA ACCGCGTCTG TTCACTCCGG AAGAATTCTT
K S F K S P E P R L F T P E E F F
CCGTATCTTC AACCGTTCCA TCGACGCTTT CAAAGACTTC GTTGTTGCTT
R I F N R S I D A F K D F V V A
CCGAAACCTC CGACTGCGTT GTTTCCTCCA CCCTGTCTCC GGAAAAAGAC
S E T S D C V V S S T L S P E K D
TCCCGTGTTT CGGTTACCAA ACCGTTCATG CTGCCGCCGG TTGCTGCTTAA
S R V S V T K P F M L P P V A A *
r The amino acid sequence and nucleic acid sequence for SCF met 1-165 N10D was (Seq. ID Nos. 3 and 4 (DNA) and 5 (amino acid)):
CATATGGAAGGTATCTGCAGGAATCGTGTGACTGACAATGTAAAAGACGTCACTAAATTG
1 ---------+---------+---------+---------+---------+---------+ 60 GTATACCTTCCATAGACGTCCTTAGCACACTGACTGTTACATTTTCTGCAGTGATTTAAC
M E G I C R N R V T ]2 N V K D V T K L
GTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTT
61 ---------+---------+---------+---------+---------+---------+ 120 CACCGTTTAGAAGGTTTTCTGATGTACTATTGGGAGTTTATACAGGGGCCCTACCTACAA
V A N L P K D Y M I T L K Y V P G M D V
TTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGAT
121 ---------+---------+---------+---------+---------+---------+ 180 AACGGTTCAGTAACAACCTATTCGCTCTACCATCATGTTAACAGTCTGTCGAACTGACTA
L P S H C W I S E M V V Q L S D S L T D
CTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAA
181 ---------+---------+---------+---------+---------+---------+ 240 GAAGACCTGTTCAAAAGTTTATAAAGACTTCCGAACTCATTAATAAGGTAGTATCTGTTT
L L D K F S N I S E G L S N Y S I I D K
CTTGTGAATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTA
241 ---------+---------+---------+---------+---------+---------+ 300 GAACACTTATATCACCTACTGGAACACCTCACGCACTTTCTTTTGP,GTAGATTCCTAGAT
L V N I V D D L V E C V K E N S S K D L
AAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTCCGTATC
301 ---------+---------+---------+---------+---------+---------+ 360 TTTTTTAGTAAGTTCTCGGGTCTTGGGTCCGAGAAATGAGGACTTCTTAAGAAGGCATAG
K K S F K S P E P R L F T P E E F F R I
TTCAACCGTTCCATCGACGCTTTCAAAGACTTCGTTGTTGCTTCCGAAACCTCCGACTGC
361 ---------+---------+---------+---------+---------+---------+ 420 AAGTTGGCAAGGTAGCTGCGAAAGTTTCTGAAGCAACAACGAAGGCTTTGGAGGCTGACG
F N R S I D A F K D F V V A S E T S D C
GTTGTTTCCTCCACCCTGTCTCCGGAAAAAGACTCCCGTGTTTCGGTTACCAAACCGTTC
421 ---------+---------+---------+---------+---------+---------+ 480 CAACAAAGGAGGTGGGACAGAGGCCTTTTTCTGAGGGCACAAAGCCAATGGTTTGGCAAG
V V S S T L S P E K D S R V S V T K P F
ATGCTGCCGCCGGTTGCTGCTTAATAGGATCC
481 ---------+---------+---------+-- 512 TACGACGGCGGCCAACGACGAATTATCCTAGG
M L P P V A A * *
The amino acid sequence and nucleic acid sequence for SCF met 1-165 N10D, N11D was (Seq. ID Nos.
6 and 7 (DNA) and 8 (amino acid)):
CATATGGAAGGTATCTGCAGGAATCGTGTGACTGACGATGTAAAAGACGTCACTAAATTG
1 ---------+---------+---------+---------+---------+---------+ 60 GTATACCTTCCATAGACGTCCTTAGCACACTGACTGCTACATTTTCTGCAGTGATTTAAC
GTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTT
61 ---------+---------+---------+--------- + 120 +---------+---------CACCGTTTAGAAGGTTTTCTGATGTACTATTGGGAGTTTATACAGGGGCCCTACCTACAA
V A N L P K D Y M I T L K Y V P G M D V -TTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGAT
121 ---------+---------+---------+---------+---------+---------+ 180 AACGGTTCAGTAACAACCTATTCGCTCTACCATCATGTTAACAGTCTGTCGAACTGACTA
L P S H C W I S E M V V Q L S D S L T D -CTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAA
181 ---------+---------+---------+---------+---------+---------+ 240 GAAGACCTGTTCAAAAGTTTATAAAGACTTCCGAACTCATTAATAAGGTAGTATCTGTTT
L L D K F S N I S E G L S N Y S I I D K -CTTGTGAATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTA
241 ---------+---------+---------+---------+---------+---------+ 300 GAACACTTATATCACCTACTGGAACACCTCACGCACTTTCTTTTGAGTAGATTCCTAGAT
L V N I V D D L V E C V K E N S S K D L -AAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTCCGTATC
301 ---------+---------+---------+---------+---------+---------+ 360 TTTTTTAGTAAGTTCTCGGGTCTTGGGTCCGAGAAATGAGGACTTCTTAAGAAGGCATAG
K K S F K S P E P R L F T P E E F F R I -TTCAACCGTTCCATCGACGCTTTCAAAGACTTCGTTGTTGCTTCCGAAACCTCCGACTGC
361 ---------+---------+---------+---------+---------+---------+ 420 AAGTTGGCAAGGTAGCTGCGAAAGTTTCTGAAGCAACAACGAAGGCTTTGGAGGCTGACG
F N R S I D A F K D F V V A S E T S D C -GTTGTTTCCTCCACCCTGTCTCCGGAAAAAGACTCCCGTGTTTCGGTTACCAAACCGTTC
421 ---------+---------+---------+---------+---------+---------+ 480 CAACAAAGGAGGTGGGACAGAGGCCTTTTTCTGAGGGCACAAAGCCAATGGTTTGGCAAG
V V S S T L S P E K D S R V S V T K P F -ATGCTGCCGCCGGTTGCTGCTTAATAGGATCC
481 ---------+---------+---------+-- 512 TACGACGGCGGCCAACGACGAATTATCCTAGG
M L P P V A A * *
6 and 7 (DNA) and 8 (amino acid)):
CATATGGAAGGTATCTGCAGGAATCGTGTGACTGACGATGTAAAAGACGTCACTAAATTG
1 ---------+---------+---------+---------+---------+---------+ 60 GTATACCTTCCATAGACGTCCTTAGCACACTGACTGCTACATTTTCTGCAGTGATTTAAC
GTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTT
61 ---------+---------+---------+--------- + 120 +---------+---------CACCGTTTAGAAGGTTTTCTGATGTACTATTGGGAGTTTATACAGGGGCCCTACCTACAA
V A N L P K D Y M I T L K Y V P G M D V -TTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGAT
121 ---------+---------+---------+---------+---------+---------+ 180 AACGGTTCAGTAACAACCTATTCGCTCTACCATCATGTTAACAGTCTGTCGAACTGACTA
L P S H C W I S E M V V Q L S D S L T D -CTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAA
181 ---------+---------+---------+---------+---------+---------+ 240 GAAGACCTGTTCAAAAGTTTATAAAGACTTCCGAACTCATTAATAAGGTAGTATCTGTTT
L L D K F S N I S E G L S N Y S I I D K -CTTGTGAATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTA
241 ---------+---------+---------+---------+---------+---------+ 300 GAACACTTATATCACCTACTGGAACACCTCACGCACTTTCTTTTGAGTAGATTCCTAGAT
L V N I V D D L V E C V K E N S S K D L -AAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTCCGTATC
301 ---------+---------+---------+---------+---------+---------+ 360 TTTTTTAGTAAGTTCTCGGGTCTTGGGTCCGAGAAATGAGGACTTCTTAAGAAGGCATAG
K K S F K S P E P R L F T P E E F F R I -TTCAACCGTTCCATCGACGCTTTCAAAGACTTCGTTGTTGCTTCCGAAACCTCCGACTGC
361 ---------+---------+---------+---------+---------+---------+ 420 AAGTTGGCAAGGTAGCTGCGAAAGTTTCTGAAGCAACAACGAAGGCTTTGGAGGCTGACG
F N R S I D A F K D F V V A S E T S D C -GTTGTTTCCTCCACCCTGTCTCCGGAAAAAGACTCCCGTGTTTCGGTTACCAAACCGTTC
421 ---------+---------+---------+---------+---------+---------+ 480 CAACAAAGGAGGTGGGACAGAGGCCTTTTTCTGAGGGCACAAAGCCAATGGTTTGGCAAG
V V S S T L S P E K D S R V S V T K P F -ATGCTGCCGCCGGTTGCTGCTTAATAGGATCC
481 ---------+---------+---------+-- 512 TACGACGGCGGCCAACGACGAATTATCCTAGG
M L P P V A A * *
Characterization Confirmation of the identity of SCF met 1-165 N10D and N10D, N11D was accomplished by N-terminal amino acid sequence of intact proteins. Amino acid sequence of SCF N10D was determined to be Met-Glu-Gly-Ile-[Cys]-Arg-Asn-Arg-Val-Thr-Asp-Asn-Val-Lys----, while the sequence of SCF N10D, N11D was elucidated to be Met-Glu-Gly-Ile-[Cys]-Arg-Asn-Arg-Val-Thr-Asp-Asp-Val-Lys----.
Therefore the determined sequences of both purified SCF
N10D and SCF N10D, N11D match the sequences predicted from the respective DNA sequences shown in SEQ ID nos. 3 and 4 and SEQ ID nos. 6 and 7.
3. Increased Biological Activity. Presented below are data demonstrating that the present recombinant human SCF analogs N10D and N10D, N11D are approximately 50% more biologically active than the non-modified recombinant human SCF 1-165.
Biological Activity Assay An in vitro UT-7 bioassay was performed by measuring the stimulatory effect of SCF on the proliferation of megakaryocytic cell culture. The 3H-thymidine uptake of the cultured cells stimulated by the samples are indicative of biological potency for SCF. Methods used were according to Smith et al., in Current Protocols in Immunology (Coligan, et al., eds.) pp. 6.17.1 to 6.17.11, John Wiley & Sons, New York.
Therefore the determined sequences of both purified SCF
N10D and SCF N10D, N11D match the sequences predicted from the respective DNA sequences shown in SEQ ID nos. 3 and 4 and SEQ ID nos. 6 and 7.
3. Increased Biological Activity. Presented below are data demonstrating that the present recombinant human SCF analogs N10D and N10D, N11D are approximately 50% more biologically active than the non-modified recombinant human SCF 1-165.
Biological Activity Assay An in vitro UT-7 bioassay was performed by measuring the stimulatory effect of SCF on the proliferation of megakaryocytic cell culture. The 3H-thymidine uptake of the cultured cells stimulated by the samples are indicative of biological potency for SCF. Methods used were according to Smith et al., in Current Protocols in Immunology (Coligan, et al., eds.) pp. 6.17.1 to 6.17.11, John Wiley & Sons, New York.
Results comparing the present analogs to unmodified SCF met 1-165 are presented below:
Sample Half Maximal % activityl stimulation concentration ( n /ml ) SCF met 1-165 5.4 100 SCF met 1-165 3.5 154 3.2 SCF met 1-165 160 N10D, N11D
1 This was determined using the half maximal stimulation concentration for standard recombinant human SCF (5.4), divided by the value for the variant (3.5 or 3.2) and multiplied by 100.
These results demonstrate that in vitro, the biological activity of the present recombinant human SCF1-165 N10D and N10D, N11D analogs is approximately 50% higher than native recombinant human SCF1-165.
4. Increased stability. Presented below are data demonstrating that the present recombinant human SCF1-165 analogs are more stable in accelerated stability assays than non-modified recombinant human SCF1-165.
Stability assays. Comparative accelerated stability among SCF, SCF N10D and SCF N10D, N11D was evaluated by incubating samples in 100 mM sodium bicrabonate, pH 8.4 at 37 oC for 1-5 days. Sulfopropyl cationic exchange HPLC using SP-5PW TSK gel columns was performed to quantify the amount of the remaining undegraded form and the deamidation products formed upon storage in the above mentioned buffer. As indicated in FIGURE 2, SCF N10D and SCF N10D, N11D are equally as stable during incubation and exhibit better stability than the standard SCF.
While the present invention has been described in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed.
Sample Half Maximal % activityl stimulation concentration ( n /ml ) SCF met 1-165 5.4 100 SCF met 1-165 3.5 154 3.2 SCF met 1-165 160 N10D, N11D
1 This was determined using the half maximal stimulation concentration for standard recombinant human SCF (5.4), divided by the value for the variant (3.5 or 3.2) and multiplied by 100.
These results demonstrate that in vitro, the biological activity of the present recombinant human SCF1-165 N10D and N10D, N11D analogs is approximately 50% higher than native recombinant human SCF1-165.
4. Increased stability. Presented below are data demonstrating that the present recombinant human SCF1-165 analogs are more stable in accelerated stability assays than non-modified recombinant human SCF1-165.
Stability assays. Comparative accelerated stability among SCF, SCF N10D and SCF N10D, N11D was evaluated by incubating samples in 100 mM sodium bicrabonate, pH 8.4 at 37 oC for 1-5 days. Sulfopropyl cationic exchange HPLC using SP-5PW TSK gel columns was performed to quantify the amount of the remaining undegraded form and the deamidation products formed upon storage in the above mentioned buffer. As indicated in FIGURE 2, SCF N10D and SCF N10D, N11D are equally as stable during incubation and exhibit better stability than the standard SCF.
While the present invention has been described in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Amgen Inc.
(ii) TITLE OF INVENTION: SCF ANALOG COMPOSITIONS AND METHODS
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amgen Inc.
(B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 91320-1789 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppv disk (B) COMPUTER: IBM PC* compatible (C) OPERATING SYSTEM: PC-DOS*/MS-DOS*
(D) SOFTUfARE: PatentIn* Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBEft: US 08/628,428 (B) FILING DATE: 05-APR-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Knight, Matthew W
(B) REGISTRATION NUMBER: 36,846 (C) REFERENCE/DOCKET NUMBER: A-400 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
* Trade-mark (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..166 (D) OTHER INFORMATION: /note= "Met sequence starts at -1 on Sequence No. 2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser - 23 _ PCT/US97/05541 Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
(1) GENERAL INFORMATION:
(i) APPLICANT: Amgen Inc.
(ii) TITLE OF INVENTION: SCF ANALOG COMPOSITIONS AND METHODS
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amgen Inc.
(B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 91320-1789 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppv disk (B) COMPUTER: IBM PC* compatible (C) OPERATING SYSTEM: PC-DOS*/MS-DOS*
(D) SOFTUfARE: PatentIn* Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBEft: US 08/628,428 (B) FILING DATE: 05-APR-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Knight, Matthew W
(B) REGISTRATION NUMBER: 36,846 (C) REFERENCE/DOCKET NUMBER: A-400 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
* Trade-mark (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..166 (D) OTHER INFORMATION: /note= "Met sequence starts at -1 on Sequence No. 2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser - 23 _ PCT/US97/05541 Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..166 (D) OTHER INFORMATION: /note= "Met sequence starts at -1 on Sequence No. 5."
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..166 (D) OTHER INFORMATION: /note= "Met sequence starts at -1 on Sequence No. 5."
- 25 _ PCTIUS97/05541 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Glu Gly Ile Cys Arg Asn Arg Val Thr Asp Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Glu Gly Ile Cys Arg Asn Arg Val Thr Asp Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AAGTTGGCAA GGTAGCTGCG AA.AGTTTCTG AAGCAACAAC GAAGGCTTTG GAGGCTGACG 420 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..166 (D) OTHER INFORMATION: /note= "Met sequence starts at -1 on Sequence No. 8."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Glu Gly Ile Cys Arg Asn Arg Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala - 28 _ PCT/US97/05541 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..273 (D) OTHER INFORMATION: /note= "NOTE: Mature full length 1-248 SCF protein begins at amino acid 26; amino acid 1-25 include Met and leader sequences for membrane band form of human recombinant SCF."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Lys Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu Phe Gln Glu Val
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AAGTTGGCAA GGTAGCTGCG AA.AGTTTCTG AAGCAACAAC GAAGGCTTTG GAGGCTGACG 420 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..166 (D) OTHER INFORMATION: /note= "Met sequence starts at -1 on Sequence No. 8."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Glu Gly Ile Cys Arg Asn Arg Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala - 28 _ PCT/US97/05541 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..273 (D) OTHER INFORMATION: /note= "NOTE: Mature full length 1-248 SCF protein begins at amino acid 26; amino acid 1-25 include Met and leader sequences for membrane band form of human recombinant SCF."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Lys Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu Phe Gln Glu Val
Claims (17)
1. An SCF analog wherein said SCF analog differs from SCF by the addition, deletion and/or substitution of at least one amino acid and is capable of stimulating the growth of early hematopoietic progenitor cells, or dimeric forms thereof, wherein said analog is an analog of a recombinant human SCF and said analog is selected from the group consisting of:
(a) any SCF analog having amino acid residues 26-163 through 26-273 of SEQ ID
NO:9, wherein each of the Asn residues at positions 35 and 36 of SEQ ID NO: 9 are replaced with an Asp residue;
(b) SCF analog having amino acid residues 26-152, 26-155, or 26-162 of SEQ ID
NO:9, wherein each of the Asn residues at positions 35 and 36 of SEQ ID NO: 9 are replaced with an Asp residue;
(c) any of the SCF analogs of subparts (a) or (b) having N-terminal deletions at the 26, 27, or 28 positions and having the cysteine remaining at position 29 of SEQ ID
NO: 9; and (d) any of the SCF analogs of subparts (a), (b), or (c) having an N-terminal methionyl residue.
(a) any SCF analog having amino acid residues 26-163 through 26-273 of SEQ ID
NO:9, wherein each of the Asn residues at positions 35 and 36 of SEQ ID NO: 9 are replaced with an Asp residue;
(b) SCF analog having amino acid residues 26-152, 26-155, or 26-162 of SEQ ID
NO:9, wherein each of the Asn residues at positions 35 and 36 of SEQ ID NO: 9 are replaced with an Asp residue;
(c) any of the SCF analogs of subparts (a) or (b) having N-terminal deletions at the 26, 27, or 28 positions and having the cysteine remaining at position 29 of SEQ ID
NO: 9; and (d) any of the SCF analogs of subparts (a), (b), or (c) having an N-terminal methionyl residue.
2. An SCF analog according to claim 1 wherein said SCF analog differs from human SCF
by the addition, deletion and/or substitution of at least one amino acid and is capable of stimulating the growth of early hematopoietic progenitor cells, or dimeric forms thereof, wherein said SCF analog has the amino acid sequence of SEQ ID NO:8.
by the addition, deletion and/or substitution of at least one amino acid and is capable of stimulating the growth of early hematopoietic progenitor cells, or dimeric forms thereof, wherein said SCF analog has the amino acid sequence of SEQ ID NO:8.
3. A DNA molecule encoding an SCF analog of claim 1 or 2.
4. A DNA molecule according to claim 3 having (a) the DNA sequence set forth in SEQ ID NO: 6 or the complementary strand thereof;
(b) a variant of the DNA sequence of subpart (a) encoding aspartic acid at position 35 and 36 of SEQ ID NO: 9.
(b) a variant of the DNA sequence of subpart (a) encoding aspartic acid at position 35 and 36 of SEQ ID NO: 9.
5. A process for producing an SCF analog according to claim 1 or 2 from a host cell containing nucleic acid encoding such SCF analog comprising the steps of:
(a) culturing said host cell containing a DNA molecule according to claim 3 or claim 4 under conditions facilitating the expression of such DNA molecule; and (b) obtaining such SCF analog.
(a) culturing said host cell containing a DNA molecule according to claim 3 or claim 4 under conditions facilitating the expression of such DNA molecule; and (b) obtaining such SCF analog.
6. A DNA viral or plasmid vector containing a DNA molecule according to claim 3 or claim 4.
7. A host cell containing a DNA of claim 3 or claim 4.
8. A host cell of claim 7 wherein said host cell is selected from the group consisting of a bacterium, a mammalian cell, a yeast cell, and an insect cell.
9. A pharmaceutical composition comprising an SCF analog according to claim 1 or 2 and a pharmaceutically acceptable diluent, adjuvant or carrier.
10. Use of an SCF analog according to claim 1 or 2 for the preparation of a medicament for treating a hematopoietic disorder.
11. Use according to claim 10, wherein said hematopoietic disorder is selected from the group consisting of leucopenia, thrombocytopenia, anemia, enhancing engraftment of bone marrow during transplantation, enhancing bone marrow recover in treatment of radiation, chemical or chemotherapeutica induced bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome.
12. Use according to claims 10 or 11, wherein the medicament includes at least one additional factor selected from among EPO, G-CSF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IGF-1, LIF, interferon, a neurotrophic factor, flt-3/flk-2 ligand, and a fibroblast growth factor.
13. A method for culturing hematopoietic cells in vitro comprising:
(i) placing said cells in a suitable culture medium, said suitable culture medium containing an SCF analog, wherein said analog is an analog according to claim or 2; and (ii) providing suitable conditions for the growth of said hematopoietic cells.
(i) placing said cells in a suitable culture medium, said suitable culture medium containing an SCF analog, wherein said analog is an analog according to claim or 2; and (ii) providing suitable conditions for the growth of said hematopoietic cells.
14. A method of transfecting hematopoietic cells with exogenous DNA
comprising:
(i) culturing said hematopoietic cells in a medium containing an SCF analog, wherein said analog is an analog according to claim 1 or 2; and (ii) transfecting said cultured cells with exogenous DNA.
comprising:
(i) culturing said hematopoietic cells in a medium containing an SCF analog, wherein said analog is an analog according to claim 1 or 2; and (ii) transfecting said cultured cells with exogenous DNA.
15. The method of claim 14 wherein said hematopoietic cells are bone marrow cells or peripheral blood progenitor cells.
16. The method of any of claims 13 through 15 wherein said culturing includes the use of at least one additional factor selected from among EPO, G-CSF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6. IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IGF-1, LIF, interferon, a neurotrophic factor, flt-3/flk-2 ligand, and a fibroblast growth factor.
17. A kit containing for culturing hematopoietic cells comprising:
(i) an SCF analog wherein said analog is an analog of claim 1 or 2;
(ii) components suitable for preparing medium for culturing one marrow cells or peripheral blood progenitor cells; and (iii) optionally, at least one additional factor selected from among EPO, G-CSF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IGF-1, LIF, interferon, a neurotrophic factor, ftt-3/flk-2 ligand, and a fibroblast growth factor.
(i) an SCF analog wherein said analog is an analog of claim 1 or 2;
(ii) components suitable for preparing medium for culturing one marrow cells or peripheral blood progenitor cells; and (iii) optionally, at least one additional factor selected from among EPO, G-CSF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IGF-1, LIF, interferon, a neurotrophic factor, ftt-3/flk-2 ligand, and a fibroblast growth factor.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/628,428 US5885962A (en) | 1996-04-05 | 1996-04-05 | Stem cell factor analog compositions and method |
| US08/628,428 | 1996-04-05 | ||
| PCT/US1997/005541 WO1997038101A1 (en) | 1996-04-05 | 1997-04-03 | Scf analog compositions and methods |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2249181A1 CA2249181A1 (en) | 1997-10-16 |
| CA2249181C true CA2249181C (en) | 2009-11-17 |
Family
ID=24518837
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002249181A Expired - Fee Related CA2249181C (en) | 1996-04-05 | 1997-04-03 | Scf analog compositions and methods |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US5885962A (en) |
| EP (1) | EP0904367B1 (en) |
| JP (1) | JP4066388B2 (en) |
| KR (1) | KR20000004951A (en) |
| CN (1) | CN1214734A (en) |
| AT (1) | ATE363488T1 (en) |
| AU (1) | AU726663B2 (en) |
| BR (1) | BR9708578A (en) |
| CA (1) | CA2249181C (en) |
| CZ (1) | CZ301598A3 (en) |
| DE (1) | DE69737766T2 (en) |
| ES (1) | ES2286830T3 (en) |
| IL (1) | IL126221A0 (en) |
| NO (1) | NO984491L (en) |
| SK (1) | SK131098A3 (en) |
| WO (1) | WO1997038101A1 (en) |
| YU (1) | YU41798A (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0789761T3 (en) * | 1994-11-04 | 2005-11-28 | Applied Research Systems | Human SCF, a splice variant thereof, its pharmaceutical use |
| US5969105A (en) * | 1996-10-25 | 1999-10-19 | Feng; Yiqing | Stem cell factor receptor agonists |
| US6967092B1 (en) | 1996-10-25 | 2005-11-22 | Mc Kearn John P | Multi-functional chimeric hematopoietic receptor agonists |
| US6291661B1 (en) | 1998-07-02 | 2001-09-18 | Immunex Corporation | flt3-L mutants and method of use |
| MXPA02000525A (en) * | 1999-07-15 | 2005-07-01 | Inst Genetics Llc | Formulations for il-11. |
| ATE480558T1 (en) * | 1999-12-23 | 2010-09-15 | Univ Geneve | SCF PEPTIDE BASED BASOLATERAL SORTING SIGNAL AND INHIBITORS THEREOF |
| AU2002362088A1 (en) * | 2001-12-06 | 2003-06-23 | Catholic University | Method and composition for inducing weight loss |
| US7439063B2 (en) * | 2002-06-11 | 2008-10-21 | Burnham Institute For Medical Research | Neuroprotective synergy of erythropoietin and insulin-like growth factors |
| US20150018408A1 (en) | 2013-07-10 | 2015-01-15 | The Regents Of The University Of Michigan | Therapeutic antibodies and uses thereof |
| CN105646709A (en) * | 2011-01-10 | 2016-06-08 | 密执安大学评议会 | Stem cell factor inhibitor |
| US20150196622A1 (en) * | 2012-08-21 | 2015-07-16 | Ali Nayer | Materials and methods for modulating glucose uptake |
| CN105985424B (en) * | 2015-01-30 | 2019-08-23 | 苏州方舟生物医药有限公司 | A kind of stem cell factor functionality peptide fragment and its application |
| WO2016187820A1 (en) * | 2015-05-26 | 2016-12-01 | 中国科学院广州生物医药与健康研究院 | Human primary cell culture medium and use thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6936694B1 (en) * | 1982-05-06 | 2005-08-30 | Intermune, Inc. | Manufacture and expression of large structural genes |
| SG43009A1 (en) * | 1989-10-16 | 1997-10-17 | Amgen Inc | Stem cell factor |
| HU220234B (en) * | 1989-10-16 | 2001-11-28 | Amgen Inc. | Process for producing factors influencing the functioning of stem cells |
| WO1992000376A1 (en) * | 1990-06-25 | 1992-01-09 | Immunex Corporation | Mast cell growth factor |
| US20030125519A1 (en) * | 1990-08-27 | 2003-07-03 | Peter Besmer | Ligand for the c-kit receptor and methods of use thereof |
| DE4224050A1 (en) * | 1991-07-31 | 1993-02-04 | Hoffmann La Roche | LOCAL KIT LIGANDS |
| US5525708A (en) * | 1994-03-28 | 1996-06-11 | Cytomed, Inc. | Covalent dimer of kit ligand |
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1996
- 1996-04-05 US US08/628,428 patent/US5885962A/en not_active Expired - Fee Related
-
1997
- 1997-04-03 IL IL12622197A patent/IL126221A0/en unknown
- 1997-04-03 ES ES97917841T patent/ES2286830T3/en not_active Expired - Lifetime
- 1997-04-03 KR KR1019980707544A patent/KR20000004951A/en not_active Application Discontinuation
- 1997-04-03 WO PCT/US1997/005541 patent/WO1997038101A1/en active IP Right Grant
- 1997-04-03 SK SK1310-98A patent/SK131098A3/en unknown
- 1997-04-03 AU AU26064/97A patent/AU726663B2/en not_active Ceased
- 1997-04-03 CA CA002249181A patent/CA2249181C/en not_active Expired - Fee Related
- 1997-04-03 EP EP97917841A patent/EP0904367B1/en not_active Expired - Lifetime
- 1997-04-03 AT AT97917841T patent/ATE363488T1/en not_active IP Right Cessation
- 1997-04-03 CN CN97193342A patent/CN1214734A/en active Pending
- 1997-04-03 DE DE69737766T patent/DE69737766T2/en not_active Expired - Fee Related
- 1997-04-03 BR BR9708578A patent/BR9708578A/en unknown
- 1997-04-03 CZ CZ983015A patent/CZ301598A3/en unknown
- 1997-04-03 JP JP53632097A patent/JP4066388B2/en not_active Expired - Fee Related
-
1998
- 1998-09-24 YU YU41798A patent/YU41798A/en unknown
- 1998-09-25 NO NO984491A patent/NO984491L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IL126221A0 (en) | 1999-05-09 |
| EP0904367B1 (en) | 2007-05-30 |
| SK131098A3 (en) | 1999-03-12 |
| EP0904367A1 (en) | 1999-03-31 |
| ATE363488T1 (en) | 2007-06-15 |
| CN1214734A (en) | 1999-04-21 |
| ES2286830T3 (en) | 2007-12-01 |
| WO1997038101A1 (en) | 1997-10-16 |
| BR9708578A (en) | 1999-08-03 |
| KR20000004951A (en) | 2000-01-25 |
| CZ301598A3 (en) | 1999-01-13 |
| JP4066388B2 (en) | 2008-03-26 |
| AU726663B2 (en) | 2000-11-16 |
| NO984491L (en) | 1998-10-05 |
| DE69737766D1 (en) | 2007-07-12 |
| CA2249181A1 (en) | 1997-10-16 |
| DE69737766T2 (en) | 2007-10-11 |
| NO984491D0 (en) | 1998-09-25 |
| AU2606497A (en) | 1997-10-29 |
| YU41798A (en) | 1999-07-28 |
| US5885962A (en) | 1999-03-23 |
| JP2000508170A (en) | 2000-07-04 |
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